The DNA replication restart pathways reload the DNA replication machinery onto replication forks that have been abandoned as a consequence of genomic damage. These pathways form an essential biochemical link between repair (often recombinational repair) of broken replication forks and DNA replication. The proteins that drive these reactions, referred to as the primosome or the Replication Restart Proteins (RRPs), must recognize the structures of these abandoned replication forks and reload the DNA replication machinery at these sites. This process is heavily regulated to ensure loading fidelity and to avoid over-replication that could arise from initiating replication at improper DNA structures. The structural mechanisms underlying DNA replication restart and the cellular mechanisms by which it is integrated with other cellular genome maintenance processes are currently poorly understood. Our proposal combines structural, biochemical, and genetic approaches to define the mechanisms of DNA replication restart pathways in complementary ways. We wil use X- ray crystallography to determine the crystal structures of key proteins and protein complexes that comprise the primosome (Aim 1). These studies will produce molecular models that will help define the physical mechanisms by which bacterial RRPs function. Additionally, we will define biochemically how RRPs interact with one another to drive replication restart (Aim 2). This set of experiments will link the physical models generated in Aim 1 to steps along the replication restart pathways, to reveal how the primosome ties replication fork recognition to RRP complex assembly. Finally, we will identify linkages that coordinate replication restart with DNA replication, recombination, and repair processes in bacterial cells (Aim 3). These connections will help define how replication restart is integrated into the basal genome maintenance network in cells and how its use is regulated to prevent unwarranted replication initiation. PUBLIC HEALTH RELEVANCE: DNA replication restart is an important process to study from a human health perspective because knowing more about DNA replication will help to identify targets to kill selectively the deleterious, malignant or disease causing cells that may plague our bodies (e.g., tumors or pathogenic organisms). Conversely, this knowledge may also be used to augment the basic health and longevity of our cells. This may be translated into a longer, healthier life for the individual.